Mladen Kerovec

Pleistocene divergence of Dinaric Drusus endemics (Trichoptera, Limnephilidae) in multiple microrefugia within the Balkan Peninsula

The Balkan Peninsula is one of three major European refugial areas. It has high biodiversity and endemism, but data on the age and origin of its fauna, especially endemics, are limited. Mitochondrial sequence data (COI and 16S genes) were used to study the population structure and phylogeography of the caddisfly Drusus croaticus and the phylogeny and divergence of seven other Drusus species, mostly range‐restricted endemics of the Dinaric region of the Balkan Peninsula. The divergence of D. croaticus populations in Croatia and allopatric Drusus species in Bosnia dated to the Pleistocene, showing the importance of this time period for the origin and diversification of Balkan endemic taxa. The divergence of more distantly related species dated to the Late Miocene/Early Pliocene. Population genetic and phylogeographic analysis of 115 individuals from 11 populations of D. croaticus revealed a high level of genetic differentiation and absence of gene flow between populations separated by more than 10 km. The existence of allopatrically fragmented lineages in D. croaticus and the endemic Bosnian species is most likely the result of long‐term isolation in multiple microrefugia, probably due to the specific habitat requirements and life‐history traits of Drusinae coupled with the topographic complexity and historical changes in geomorphology of the region. Overall, these findings shed light on the processes generating the high genetic complexity of this refugial region that parallels the ‘refugia within refugia’ pattern widely reported from the Iberian refugium.

Microscale vicariance and diversification of Western Balkan caddisflies linked to karstification

Abstract: The karst areas in the Dinaric region of the Western Balkan Peninsula are a hotspot of freshwater biodiversity. Many investigators have examined diversification of the subterranean freshwater fauna in these karst systems. However, diversification of surface-water fauna remains largely unexplored. We assessed local and regional diversification of surface-water species in karst systems and asked whether patterns of population differentiation could be explained by dispersal—diversification processes or allopatric diversification following karstrelated microscale vicariance. We analyzed mitochondrial cytochrome c oxidase subunit I (mtCOI) sequence data of 4 caddisfly species (genus Drusus) in a phylogeographic framework to assess local and regional population genetic structure and Pliocene/Pleistocene history. We used BEAST software to assess the timing of intraspecific diversification of the target species. We compared climate envelopes of the study species and projected climatically suitable areas during the last glacial maximum (LGM) to assess differences in the species climatic niches and infer potential LGM refugia. The haplotype distribution of the 4 species (324 individuals from 32 populations) was characterized by strong genetic differentiation with few haplotypes shared among populations (16%) and deep divergence among populations of the 3 endemic species, even at local scales. Divergence among local populations of endemics often exceeded divergence among regional and continental clades of the widespread D. discolor. Major divergences among regional populations dated to 2.0 to 0.5 Mya. Species distribution model projections and genetic structure suggest that the endemic species persisted in situ and diversified locally throughout multiple Pleistocene climate cycles. The pattern for D. discolor was different and consistent with multiple invasions into the region. Patterns of population genetic structure and diversification were similar for the 3 regional endemic Drusus species and consistent with microscale vicariance after the onset of intensified karstification in the Dinaric region. Karstification may induce microscale vicariance of running surface-water habitats and probably promotes allopatric fragmentation of stream insects at small spatial scales.

Estimation of DNA Integrity in Blood Cells of Eastern Mosquitofish (Gambusia holbrooki) Inhabiting an Aluminium-Polluted Water Environment: an Alkaline Comet Assay Study

To estimate the impacts of an Al-contaminated aquatic environment on DNA integrity in the blood cells of eastern mosquitofish Gambusia holbrooki Girard 1859 inhabiting Lake Njivice (Island of Krk, Croatia), an evaluation using the alkaline comet assay was carried out. Genome integrity was studied in parallel with the same fish species inhabiting the nearby, unpolluted Lake Ponikve. The amount of DNA damage in cells was estimated from three different parameters: comet tail length as the extent of genetic material migration, tail intensity (% DNA in the comet tail) and tail moment. The results indicate the loss of genome integrity in blood cells of mosquitofish inhabiting Lake Njivice and the genotoxicity of this aquatic environment. Using the same assay, acute genotoxicity of contaminated water and sediment was evaluated and confirmed on fish, mouse and human blood cells treated ex vivo. Results of the present study indicate that the alkaline comet assay applied to fish blood cells is a valuable tool for determining the potential genotoxicity of water pollutants and confirm its usefulness in the evaluation of DNA damage in fish living in Al-polluted waters.

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: A case study

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.

A cave leech (Hirudinea, Erpobdellidae) from Croatia with unique morphological features

Croatobranchus mestrovi is a troglobitic leech from deep shaft‐like caves in the Velebit Mountain, Dinaric karst, Croatia, living in cold (4–6 °C) water. Its oral sucker extends to form four pairs of triangular tentacles, each with about five finger‐like papillae, but widening into a marginally crenulated disc when attached to a substrate. Pairs of stiff, finger‐like lateral projections, probably gills, occur along the body behind the clitellum. Somites are simple five‐annulate. Despite the unique head morphology and the presence of lateral outgrowths, the anatomy and 18S rRNA gene sequence of this species indicate that it is a member of the Erpobdellidae, closely related to Dina.

Crustacean plankton community (Crustacea: Copepoda and Cladocera) in gypsum karst lakes and their relation to abiotic parameters

Two gypsum karst lakes and one non-gypsum karst lake were studied for copepod and cladoceran species composition in relation to specific habitat characteristics. The investigation was conducted from March to June 2007. Four copepod and six cladoceran species were recorded. Gypsum karst lakes are characterized as sulphate lakes and they show significant differences from non-gypsum karst lakes in conductivity, TDS, alkalinity, calcium and sulphates. Data on environmental variables and zooplankton were analyzed using redundancy analysis (RDA). The model explained 65.73% of the variance of the crustacean zooplankton and environmental data by the first two axes. The analysis confirmed that the major environmental variables influencing zooplankton in gypsum karst lakes are conductivity, TDS, calcium and sulphates. In the non-gypsum karst lake, on the contrary, the major variables were oxygen concentration and alkalinity. Specific habitat characteristics of gypsum karst lakes influence the zooplankton community by reducing the number of species and leading to the dominance of one of them.

Copepod communities in karstic mediterranean lakes along the eastern Adriatic coast

The copepod communities of karstic lakes along the eastern Adriatic coast were studied. Lakes were divided in several groups according to their morphology (deep, shallow, barrage and reservoirs), production (oligotrophic, mesotophic and eutrophic), and salinity of water: freshwater and brackish. Copidodiaptomus steueri, Eucyclops serrulatusand Macrocyclops albidus belong to the group that inhabited most of the lakes under the study, regardless of lake type. The shallow karstic lakes are usually inhabited by Thermocyclops dybowskii, T. oithonoides, Cyclops vicinus and Eudiaptomus padanus etruscus in freshwater biotopes, and Calanipedia aquaedulcis and Copidodiaptomus steueri in brackish biotopes. The last two species can also be found in deep karstic lakes with brackish water (Bacina lakes in the Neretva River delta). Species like Cyclops abyssorumcan be found in most deep freshwater lakes. Some Calanoida were recorded in only one lake, like Eudiaptomus transsylvanicusin the deep Lake Vrana on the island of Cres, or Eudiaptomus hadzici in the barrage Lake Visovac. Production of the lakes, expressed as copepod biomass, depends on lake trophy, and in some lakes also on hydrology and salinity. Most of the meso-eutrophic lakes in the study area had Calanoida dominating, while Cyclopoida dominated in some oligotrophic and eutrophic lakes as well.

Ecologically acceptable flows definition for the Žrnovnica river (Croatia)

The paper defines an ecologically acceptable flow regime for the River Žrnovnica, Croatia. This is a small and relatively short karst river with high flows and high quality water, convenient for use for public water supply. Because the water from the river will be taken from the karst spring zone, the entire river channel could suffer negative ecological consequences. The main goal of setting minimum acceptable flows is to protect the Žrnovnica river food webs and to sustain the rare and endangered fish species Salmothymus obtusirostris. This paper presents results obtained from hydrological, morphological and biological investigations.

Spatial distribution and seasonal changes of mayflies (Insecta, Ephemeroptera) in a Western Balkan peat bog

Abstract Peat bogs are unique wetland ecosystems of high conservation value all over the world, yet data on the macroinvertebrates (including mayfly assemblages) in these habitats are still scarce. Over the course of one growing season, mayfly assemblages were sampled each month, along with other macroinvertebrates, in the largest and oldest Croatian peat bog and an adjacent stream. In total, ten mayfly species were recorded: two species in low abundance in the peat bog, and nine species in significantly higher abundance in the stream. Low species richness and abundance in the peat bog were most likely related to the harsh environmental conditions and mayfly habitat preferences. In comparison, due to the more favourable habitat conditions, higher species richness and abundance were observed in the nearby stream. Three of the recorded species, Caenis luctuosa from the peat bog, and Eurylophella karelica and Leptophlebia marginata from the stream are new records for the Croatian mayfly fauna. Typical Central European life cycle patterns were confirmed for several species (e.g. Baetis vernus, Nigrobaetis niger, Electrogena ujhelyii), while for several others (e.g. Habrophlebia fusca, Paraleptophlebia submarginata) some discrepancies were observed. Therefore, these results provide new and valuable information on the ecology of mayflies in peat bog habitats.

Copepod communities in karstic mediterraneanlakes along the eastern Adriatic coast

The copepod communities of karstic lakes along the eastern Adriatic coast were studied. Lakes were divided in several groups according to their morphology (deep, shallow, barrage and reservoirs), production (oligotrophic, mesotophic and eutrophic), and salinity of water: freshwater and brackish. Copidodiaptomus steueri, Eucyclops serrulatus and Macrocyclops albidus belong to the group that inhabited most of the lakes under the study, regardless of lake type. The shallow karstic lakes are usually inhabited by Thermocyclops dybowskii, T. oithonoides, Cyclops vicinus and Eudiaptomus padanus etruscus in freshwater biotopes, and Calanipedia aquaedulcis and Copidodiaptomus steueri in brackish biotopes. The last two species can also be found in deep karstic lakes with brackish water (Bacina lakes in the Neretva River delta). Species like Cyclops abyssorum can be found in most deep freshwater lakes. Some Calanoida were recorded in only one lake, like Eudiaptomus transsylvanicus in the deep Lake Vrana on the island of Cres, or Eudiaptomus hadzici in the barrage Lake Visovac. Production of the lakes, expressed as copepod biomass, depends on lake trophy, and in some lakes also on hydrology and salinity. Most of the mesoeutrophic lakes in the study area had Calanoida dominating, while Cyclopoida dominated in some oligotrophic and eutrophic lakes as well.